We have continued to explore the role that reactive oxygen species (ROS)play in signal transduction pathways. We have previously shown that a variety of growth factors and cytokines induce the generation of ROS following ligand binding. Our studies suggest that the pathway leading to ROS generation involves the activation of the small GTP-binding proteins rac1. This has led us to explore the role of this family of proteins in redox regulation. We have also explored the role that ROS play in apoptosis, and more recently in replicative senescence and aging. In the last year we have been able to demonstrate that oxidants function as specific regulators of pathways linked to aging. In particular, we have demonstrated a relationship between forkhead proteins, a gene family that regulates longevity in C. elegans, p66shc the only gene known to regulate longevity in mammalian cells, and intracellular ROS, thought to regulate aging across a diverse number of species. These studies published in the widely read journal Science, provided a mechanistic link between oxidant signal transduction and gene products linked to longevity. We are continuing to pursue these results attempting to understand whether other gene products implicated in longevity are part of this signaling cascade. We have also derived a genomic screening strategy to isolate gene products that regulate mitochondrial oxidant production and potentially regulate cellular and organismal life span. This has led to candidate gene identification of proteins that can positively or negatively regulate lifespan. We are currently testing these candidate genes in a number of assay systems from cell culture models of senescence to life span assays in simple organisms such as C.elegans. We are also exploring the role of uncoupling proteins in mitochondrial function. We have also begun to explore the field of energy signal tranduction, and whether intracellular ROS function as messengers between the mitochondria and the rest of the cell. In particular we have begun to study regulators of mitochondrial number such as the transcriptional coactivator, PGC-1. We have recently isolated a protein partner of PGC-1 that appears to inhibit the activity of the coactivator. We are pursuing these observations with a number of in vitro and in vivo studies. Finally, we are also directly assessing the in vivo role of a number of classic oxidases. Studies are now underway to genetically disrupt the xanthine oxidase gene to further understand the role of this oxidase in normal and pathophysiological conditions. We believe that this gene product may be involved in regulating blood pressure as well as regulating tissue damage secondary to conditions such as myocardial infarction or stroke.